Several mammalian proteins important for analysis of in vitro DNA replication have been purified and characterized. These proteins include two DNA alpha-polymerases, DNA beta-polymerase, and helix-destabilizing proteins from a mouse tissue and also vaccinia virus-induced DNA polymerase and vaccinia virus-induced primase from vaccinia virus-infected HeLa cells. The subunit composition of each of the mouse DNA polymerases was determined. Tryptic peptide mapping revealed that the alpha-polymerase and beta-polymerase do not have a common subunit or detectable region primary structure homology. DNA alpha 1-polymerase and vaccinia virus-induced DNA polymerase have associated exonuclease. Three discrete fragments of helix-destabilizing protein were produced by controlled proteolysis with trypsin. These fragments have been purified, and they are being used in studies on structure-function relationship of helix-destabilizing protein.